5.1 – section 5.1.1 5.1.1 introductionwolfson.huji.ac.il/purification/pdf/others/pall/pall... ·...

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5.1.1 Introduction

Today’s research and diagnostic laboratories require purified water that does not impact testresults. As testing equipment becomes more sensitive and industry regulations becomemore stringent, life science and analytical labs must have complete confidence that theirpoint-of-use water systems will provide them optimum water quality—every time.Cascada™ Lab Water Systems employ Pall’s advanced water purification technologies toachieve the highest purity water through a series of separation and purification steps in asingle bench top box. A range of configurations support diverse laboratory needs fromcritical, sensitive applications to general use.

Cascada systems offer a multitude of benefits including multi-stage monitoring and “real-time” Total Organic Carbon (TOC) monitoring. The systems can be comprehensively andcompletely sanitized. Each unit's cartridges have complete traceability.

For sensitive applications that require high quality, ultrapure water, choose a customizedpolishing system:• Cascada BIO-water System — a polishing system for demanding life sciences

applications such as cell culture, in vitro fertilization, and microfluidic and array systems.• Cascada AN-water System — a polisher for demanding analytical applications such as

ultra-trace environmental analysis.

For less demanding applications, choose one of these polishing systems:• Cascada IX-water System — a polishing system for less demanding lab water

applications, including analytical sample preparation.• Cascada LS-water System — a polisher for general-purpose lab analysis including less

sensitive life science applications.

The Cascada RO-water System is a pre-treatment system that protects and extends the lifeof the polishers. This is particularly important in cases where there is poor feed water quality.

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5.1.2 Cascada™ BIO-Water System

The Cascada BIO-water System provides extremely high quality water for life sciencesapplications by featuring ultrafiltration for the effective removal of contaminants such asendotoxins and nucleases. Endotoxin removal makes the purified water suitable for criticalapplications in cell culture and monoclonal antibody production. Nuclease eliminationremoves the requirement for toxic DEPC treatment in applications such as PCR and DNAsequencing. The use of a dual wavelength UV photo-oxidation technology at 185 and 254 nm generates hydroxyl radicals (OH) via ozone and singlet oxygen. The hydroxyl radicalhas a very high oxidation potential (2.80 volts) and is a key species in the oxidation processof organic molecules. Organic molecules are oxidized to charged organic acids and to CO2

that combine with water to generate carbonate and bicarbonate ions. All these newlyformed ions are retained by mixed ion exchange resins placed downstream from the UVlamp. In addition, these UV-C wavelengths are germicidal, killing bacteria by causingdamage to their nuclear DNA, thus preventing cell division.

BIO-water Purification System FlowPre-treated water enters the system (for fluid flow diagram, see Figure 5.1) and is pumpedthrough the primary purification pack. The first purification pack removes most of theimpurities from the water and the intermediate water quality sensor then measures theresistivity of the water from the first pack to determine when it needs to be replaced.Purified water flows directly through the UV chamber, where it is exposed to intense UVradiation at wavelengths of 254 and 185 nm to provide continuous bacterial control andphoto-oxidation of residual organic impurities. The second temperature-compensatedquality sensor provides data for TOC monitoring. Any remaining ionic and organic impuritiesare removed by the second polishing purification pack. The ultrafilter removes pyrogens,bacteria, and other microbial impurities as well as particles. Final water resistivity andtemperature are measured before dispensing. Water within the unit is recirculated throughthe purification system to maintain purity. An intermittent recirculation can be used overnightin ‘sleep mode.’ An optional 0.2 µm point-of-use (POU) filter is available if required toprotect the outlet from bacterial contamination. The specifications of the Cascada BIO-water System are summarized in Table 5.1.

Figure 5.1Fluid Flow in Cascada BIO-Water System

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Table 5.1Specifications of Cascada™ BIO-Water System

Specification Parameter

Resistivity at 25 °C 18.2 MΩ-cm

TOC (RO Pre-treatment) < 3 ppb*

Bacteria < 1 CFU/mL

Bacterial Endotoxin < 0.001 EU/mL

Particles Ultrafilter

Flow Rates Up to 2 L/minute

Dual Purification Cartridges Yes

Photo-oxidation 185/254 nm UV

0.2 µm Optional POU Filter Yes

Recirculation Yes

Interstage Water Purity Monitoring Yes

Product Water Purity Monitoring Yes

Real Time TOC Monitoring Yes

*Dependent on feed water. Recommended feed < 50 ppb TOC.

Application Data for Cascada BIO-Water System

18.2 Megohm.cm (MΩ-cm) ultrapure water for critical laboratory applications:

• Cell culture— Culture media preparation

• Reagent preparation— Buffers for purification by HPLC— Buffers for dialysis

• Microbiology— Culture media preparation

• Electrophoresis— Running buffer— Silver stain reagents

• Monoclonal antibody production• Molecular biology

— PCR— DNA sequencing

• In vitro fertilization

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Ordering Information for Cascada™ BIO-Water System

Part Number Description Pkg

PAL-CAXXBIOM2 Cascada BIO-water System 1/pkg

PAL-C180 RO Feed (DT) Cartridge Pack 1/pkg

PAL-C181 SDI Feed (DT) Cartridge Pack 1/pkg

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5.1.3 Cascada™ AN-Water System

The Cascada AN-water System provides ultrapure water for optimal chromatographicresults and longer column life by incorporating UV photo-oxidation technology for oxidationof organic contaminants at 185 nm and 254 nm generating hydroxyl radicals (OH) via ozoneand singlet oxygen. The hydroxyl radical has a very high oxidation potential (2.80 volts) andis a key species in the oxidation process of organic molecules. Organic molecules areoxidized to charged organic acids and to CO2, which combines with water to generatecarbonate and bicarbonate ions. All these newly formed ions are retained by mixed ionexchange resins placed downstream from the UV lamp. In addition, these UV-Cwavelengths are germicidal, killing bacteria by causing damage to their nuclear DNA, thuspreventing cell division. The ultra-microfiltration filter removes colloids and bacteria. The finalpurified water has extremely low levels of inorganic and organic contaminants for the mostdemanding high-performance liquid chromatography (HPLC), gas chromatography massspectrometry (GC-MS), ion chromatography, solid phase extraction, and other UVspectrometry applications.

AN-water Purification System FlowPre-treated water enters the system (for fluid flow, see Figure 5.2) and is pumped throughthe primary purification pack. The first purification pack removes most of the impurities fromthe water and the intermediate water quality sensor then measures the resistivity of thewater from the first pack to determine when it needs to be replaced. Purified water flowsdirectly through the UV chamber where it is exposed to intense UV radiation at wavelengthsof 254 and 185 nm to provide continuous bacterial control and photo-oxidation of residualorganic impurities. The second temperature-compensated quality sensor provides data forTOC monitoring. Any remaining ionic and organic impurities are removed by the secondpolishing purification pack. The 0.05 µm ultra-microfilter removes bacterial impurities as wellas particles. Final water resistivity and temperature are measured before dispensing. Waterwithin the unit is re-circulated through the purification system to maintain purity. Anintermittent recirculation can be used overnight in ‘sleep mode.’ An optional 0.2 µm point-of-use (POU) filter is available if required to protect the outlet from bacterial contamination.The specifications of the Cascada AN-water System are summarized in Table 5.2.

Figure 5.2Fluid Flow in Cascada AN-Water System

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Table 5.2Specifications of Cascada™ AN-Water System




Bacteria < 1 CFU/mL

Particles 0.05 µm ultra-microfilter

Flow Rates Up to 2 L/minute

Dual Purification Cartridges Yes


0.2 µm Optional POU Filter Yes

Recirculation Yes

Interstage Water Purity Monitoring Yes


Real Time TOC Monitoring Yes


Application Data for Cascada AN-Water System

18.2 Megohm.cm (MΩ-cm) ultrapure water for critical laboratory applications:

• Ultra-trace and trace inorganic and organic analysis• HPLC• GC-MS• ICP-MS• CF-AAS• TOC analysis• Ion chromatography• Solid phase extraction• Electrochemistry

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Ordering Information for Cascada™ AN-Water System


PAL-CAXXXANM2 Cascada AN-water System 1/pkg

PAL-C180 RO Feed (DT) Cartridge Pack 1/pkg

PAL-C181 SDI Feed (DT) Cartridge Pack 1/pkg

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5.1.4 Cascada™ IX-Water System

Pre-treated water enters (for fluid flow diagram, see Figure 5.3) via an inlet solenoid and isthen pumped through the polishing purification pack and temperature and water qualitysensors before being dispensed or re-circulated through a non-return valve back to thepump inlet. Ionic and organic impurities are removed by the polishing purification pack.Product water resistivity and temperature are measured before dispensing and will indicatewhen the purification pack needs to be replaced. The water within the unit is recirculatedthrough the purification unit to maintain purity. To reduce heat build up, the recirculation is ata reduced flow rate and is set to be intermittent (5 minutes every hour). A point-of-use filteris required to protect the outlet from bacterial contamination. The specifications of theCascada IX-water System are summarized in Table 5.3.

Figure 5.3Fluid Flow in Cascada IX-Water System

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Table 5.3Specifications of Cascada™ IX-Water System




Bacteria < 1 CFU/mL

Particles 0.2 µm Point-of-use filter

Flow Rates 1.5 L/minute

Single Purification Cartridge Pack Yes

Photo-oxidation No

0.2 µm POU Filter Required

Recirculation Yes

Product Water Temperature Monitoring Yes


Consumable Change Reminders Yes


Application Data for Cascada IX-Water System

Designed for general laboratory applications. Removes ionic and organic contaminants totrace levels. Use the 0.2 µm point-of-use (POU) filter for removal of bacteria and particlesgreater than 0.2 µm.

Ordering Information for Cascada IX-Water System


PAL-CAXXXIXM2 Cascada IX-water system 1/pkg

PAL-C195 Purification (DT) Cartridge Pack 1/pkg

PAL-C167 Point-of-Use Filter 1/pkg

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5.1.5 Cascada™ LS-Water System

Pre-treated water enters (for fluid flow diagram, see Figure 5.4) via an inlet solenoid and isthen pumped through the UV chamber, a polishing purification pack, an ultra-filter, andtemperature and water quality sensors before being dispensed or re-circulated through anon-return valve back to the pump inlet.

Purified water flows directly through the UV chamber where it is exposed to intense UVradiation at wavelengths of 254 nm and 185 nm to provide continuous bacterial control andthe photo-oxidation of residual organic impurities. Ionic and organic impurities are removedby the polishing purification pack. The ultra-filter removes pyrogens, bacteria, and othermicrobial impurities as well as particles.

Product water resistivity and temperature are measured before dispense and will indicatewhen the purification pack needs to be replaced. The water within the unit is recirculatedthrough the purification units to maintain purity. To reduce heat build up, the recirculation is ata reduced flow rate and is set to be intermittent (5 minutes every hour). The specifications ofthe Cascada LS-water System are summarized in Table 5.4.

Figure 5.4Fluid Flow in Cascada LS-Water System

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Table 5.4Specifications of Cascada™ LS-Water System




Bacteria < 1 CFU/mL

Endotoxin < 0.005 EU/mL

Particles Ultrafilter

Flow Rates 1.2 L/minute

Single Purification Cartridge Pack Yes


0.2 µm POU Filter Optional

Recirculation Yes

Product Water Temperature Monitoring Yes


Consumable Change Reminders Yes


Application Data for Cascada LS-Water System

18.2 Megohm/cm (MΩ-cm) ultrapure water for critical laboratory applications:

• ELISA tests• Affinity chromatography• Electrophoresis• Media preparation• Nucleic acid and protein isolation• Monoclonal antibody production• Mass spectrometry

Ordering Information for Cascada LS-Water System


PAL-CAXXXLSM2 Cascada LS-water System 1/pkg

PAL-C195 Purification (DT) Cartridge Pack 1/pkg

PAL-C167 Point-of-Use Filter 1/pkg

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5.1.6 Cascada™ RO-Water System

Potable water enters through a strainer (for fluid flow diagram, see Figure 5.5) and passesthrough the pre-treatment cartridge. The pre-treatment cartridge has been designed toprotect the reverse osmosis cartridges from particulate/colloidal matter and excessive freechlorine that may be present in the incoming feed water. The water passes the sanitizationport and moves through two reverse osmosis cartridges, set up in series, which split theflow into permeate and concentrate streams. The concentrate from cartridge 1 is cycled tothe inlet of cartridge 2. The resulting permeate streams are combined and pass through thewater quality sensor, which measures the conductivity of the water, and the temperaturesensor, which provides accurate temperature measurement. The final concentrate fromcartridge 2 is passed to waste.

The resulting purified water is delivered to a treated water reservoir where water quality ismaintained at the highest levels, using a built-in, automated auto-rinse cycle. This cycle isperformed each time the process is initiated and consists of a 1 minute high flow rinseacross the RO cartridges to the drain. The specifications of the Cascada RO-water Systemare summarized in Table 5.5.

Figure 5.5Fluid Flow in Cascada RO-Water System

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Table 5.5Specifications of Cascada™ RO-Water System


Inorganics – Typical Up to 98% rejection

Organics (MW > 100 Dalton) > 99% rejection

Total Organic Carbon (TOC) – < 0.1 ppmTypical (Dependent on Feed Water)

Bacteria < 5 CFU/mL

Particles > 99% rejection

Flow Rates 15 L/hour

Power Fail-Safe Yes

Automatic Level Controls Yes

Audiovisual Alarm Settings Yes - selectable

Auto-restart Yes

RO Permeate Purity Monitoring Yes in µS/cm

RO Permeate Temperature Monitoring Yes in °C

Reservoir Level Monitoring Yes % full

Consumable Remaining Life Indicator Yes

Application Data for Cascada RO-Water System

• Generation of Type III primary-grade water from a potable tap feed to supply otherultrapure water purification systems

• Routine glassware washing• Hydroponics• Feed water for humidifiers and autoclaves• Use in environmental cabinets and stills• Steam generators

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Ordering Information for Cascada™ RO-Water System


PAL-CAXXXROM2 Cascada RO-water System 1/pkg

PAL-A653 Docking Vessel-35 liter 1/pkg

Optional accessories to complement the Cascada RO-water System include printer kit,boost pump, and water storage reservoirs.

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5.2.1 Monitoring for Bacteriological Contamination with MicroFunnel™ Plus Filter Funnels

Membrane filtration (MF) is widely accepted throughout the world for the analysis ofmicrobial contamination in fluid samples. Using the MF technique, microorganisms arecaptured on a microporous membrane filter surface and grown into countable colonies byincubation of the filter with a suitable growth medium. Pall Life Sciences has designed theMicroFunnel Plus filter funnel (see Figure 5.6) to simplify this testing process. This filterfunnel can be used in accordance with the MF procedures referenced in Standard Methodsfor the Examination of Water and Wastewater, 20th edition, and the U.S. EnvironmentalProtection Agency’s Microbiological Methods for Monitoring the Environment, 600/8-78-017. MicroFunnel filter funnels also meet the USP-24 requirements for products used forthe MF technique.

Figure 5.6MicroFunnel Filter Funnels for Microbiology Testing

The MicroFunnel Plus filter funnel is supplied pre-assembled and sterilized by gammairradiation to eliminate the potential for toxic residuals associated with EtO sterilization. Theintegral unit and membrane ensure high bacterial recoveries and no grid line inhibition. Aftercapture of the microorganisms, the membrane is easily removed from the base via a uniquesqueeze separation process, without interfering with the membrane. This greatly simplifiestransfer to the growth media-soaked absorbent pad incorporated into the cap. The fullspecifications of the MicroFunnel Plus system are summarized in Table 5.6. Threemembrane types are available: • GN-6 Metricel® membrane: a 0.45 µm mixed cellulose ester membrane ideal for use in

most applications.• Supor® membrane: a 0.2 µm polyethersulfone membrane ideal for its low binding

characteristics and superior retention of small organisms such as Pseudomonas sp.• Metricel Black membrane: a 0.45 µm modified polyethersulfone membrane ideal for yeast

and mold analysis or providing good contrast for light-colored colonies.

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Table 5.6Specifications for MicroFunnel™ Plus Microbiology Testing Device

Parameter

Specification MicroFunnel Plus 100 mL MicroFunnel Plus 300 mL

Membrane 0.45 µm GN-6 Metricel® mixed cellulose ester membrane0.2 µm Supor® polyethersulfone membrane

Black 0.45 µm Metricel modified polyethersulfone membrane

Device Support Pad CelluloseCylinder Base and Petri Dish Lid (LP) PolypropyleneCover PolysyrenePlug PolyethyleneFunnel Adapter Polyethylene (not autoclavable)

Effective Filtration Area 13.46 cm2

DimensionsHeight (with Lid Petri Dish) 8.05 cm (3.17 in.) NAHeight with Cover 7.62 cm (3.00 in.) 9.09 cm (3.58 in.)Diameter(with Lid Petri Dish) 6.40 cm (2.52 in.) NADiameter with Cover 6.05 cm (2.38 in.) 8.84 cm (3.48 in.)

Capacities 100 mL 300 mL

Operating Vacuum 635 mm Hg (25 in. Hg)

Sterilization Individually packed and sterilized by gamma irradiation


5.2 – Section 5.2.1

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In this section, protocols will be described for use of MicroFunnel™ Plus disposable devicesfor testing aqueous samples for microbial contamination. Culture media ampoules areavailable for a range of bacterial, yeast, and fungal contaminants and are summarized in aproduct selection guide in Table 5.7.

Table 5.7Application Selection Guide for MicroFunnel Plus

Test Method Method Number* Recommended Filter Media Broth

Total Coliforms 9222 GN-6 Metricel® MF-Endo

Fecal Coliforms, E. coli 9222 GN-6 Metricel M-FC

Total Bacteria 9215; USP 61; 71 GN-6 Metricel, Supor® M-TGE; TSB-USP; HPC

Lactobacillus sp. detection — GN-6 Metricel Orange Serum

Yeast and Mold 9610 GN-6 Metricel, Black Supor M-Green YM

Fecal Streptococcus 9230 GN-6 Metricel KF-Streptococcal

Pseudomonas sp. 9213 Supor Pseudomonas

*Standard Methods for the Examination of Water and Wastewater, 19th ed. and U.S. Pharmacopoeia XXIV

Protocol for Monitoring for Bacteriological Contamination with MicroFunnel Plus Filter Funnels

A. Materials Required1. MicroFunnel Plus pre-sterilized devices in 100 and 300 mL volume sizes

a) 0.45 µm GN-6 Metricel membrane, griddedb) 0.2 µm Supor membranec) Metricel Black modified polyethersulfone membrane

2. MicroFunnel Plus LP (PN 4810) with 0.45 µm GN-6 Metricel membrane, white,gridded, sterile with absorbent pad incorporated into the cap for the funnel, 50/pkg

3. Selective growth media ampoules (2 mL volume)4. Sterile petri dishes and absorbent pad (PN 7245)5. Sterile phosphate buffered saline (PBS)6. Stainless steel forceps (PN 51147)7. Filter funnel manifold, single place in aluminum (PN 15408)8. Hand operated vacuum pump (PN 4041) and filtering flask with sidearm (PN 4040)9. Source of vacuum [63.5 cm Hg (25 in. Hg)] fitted with an in-line VacuShield

hydrophobic PTFE filter (PN 4402)10. Microbiological incubator

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B. Operation of the MicroFunnel™ Plus Device to Collect Microorganisms andGrowth Inside the Same DeviceIn this next section, handling of the above funnel system will require the use of aseptictechniques to avoid environmental contamination of the sample to be tested. Thismanual assumes that the operator is aware of basic sterile techniques which will not becovered in detail in the following protocols. There are several culture media ampoulesavailable for a range of bacterial, yeast, and fungal contaminants summarized in aproduct selection guide in Table 5.7. It may be necessary to process several parallelsamples in different culture media to cover bacterial, yeast, and fungal contaminants.1. Carefully remove the vented lid under aseptic or clean conditions to protect the

sterility of the MicroFunnel device.2. Collect the sample and introduce into the open device with a sterile or clean

measuring cylinder. Make a notation of the sample volume.3. Snap the vented lid back in place and seal the device for transport to the

microbiology lab for testing. Label device to identify the sample.4. Before mounting on the filtration manifold (PN 15408), remove the plug or vent tab

from the base of the device. Place on manifold and draw liquid sample through themembrane filter.

5. Wash the membrane surface with 2 x 25 mL of sterile PBS. Tip: This step may require more than 2 x 25 mL of sterile PBS, depending on whether thesample matrix is more complex than a water sample. It is important to remove components ofthe original sample that might inhibit rapid growth on the specific culture media. Organismsmay be present in the sample and only growing very slowly.

6. Gently grasp the funnel and remove and discard the lid (see Figure 5.7).Figure 5.7Separation of the Funnel from the Base of the MicroFunnel Plus Filter Funnel

7. Release funnel cylinder from base by firmly squeezing the midpoint of the cylinder.8. Culture the samples in the MicroFunnel filter funnel base by gently dispensing 2 mL

of culture media over the surface sample and allowing it to be drawn into the pad.It may be necessary to momentarily apply vacuum just until the media is drawnthrough the membrane into the pad.

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9. Replace the cover onto the base of the MicroFunnel™ unit to convert into a Petridish and discard the cylinder.

C. Culture of the Microorganisms on the Filter in a Petri Plate1. Soak adsorbent pad in sterile petri dish (PN 7245) with appropriate selective

growth media.2. Remove filter from the MicroFunnel base with sterilized forceps and carefully place

on the absorbent pad in the petri dish.3. Replace the cap on the petri dish, invert, and incubate at 37 ºC till colonies appear

on the gridded membrane (see Figure 5.8).Figure 5.8Addition of Growth Medium to the Cap of the MicroFunnel Plus LP Device

D. Culture of the Microorganisms in the Cap of a MicroFunnel Plus LP1. Dispense the contents of an ampoule of culture medium onto the absorbent pad in

the Petri dish lid kit (supplied only with MicroFunnel LP, PN 4810).2. Following the procedure described in Step 1.2.7 and remove the membrane filter

from the base with forceps.3. Place the membrane filter onto the broth-soaked absorbent pad in the petri dish lid

kit. Cover, invert, and incubate.E. Interpretation of the Results

1. After incubation for 24-48 hours (depending on media used), single isolatedcolonies of microorganism should be visible on the plates (see Figure 5.9).

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Figure 5.9Examples of Microorganisms Cultured on the Membranes in a MicroFunnel™ Plus Device

2. Count the number of colonies by placing on a light box to backlight the plates.Calculate the number of organisms per unit volume of sample.

Tip: Duplicate samples should be run to allow for uneven distribution of the contaminatingmicroorganism in the original sample.

3. If a large number of colonies are seen that are too dense to count, it may benecessary to repeat the study with an original sample diluted with sterile PBS tolower the number of organisms cultured on the filter.

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5.2 – Section 5.2.1

Pseudomonas BrothPseudomonas Broth

MF-Endo Broth

M-Green YM Broth

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Ordering Information for Monitoring for Bacteriological Contamination with MicroFunnel™ PlusFilter Funnels

Microfunnel Filter Funnels


4807* 0.45 µm GN-6 Metricel® membrane, white, gridded, sterile, 50/pkgindividually bagged, 100 mL capacity

4801 LP unit with 0.45 µm GN-6 Metricel membrane, white, 50/pkggridded, sterile, individually bagged, Petri dish lid

4808* 0.45 µm Metricel Black membrane, gridded, sterile, 50/pkgindividually bagged,100 mL capacity

4809 0.2 µm Supor® membrane, white, gridded, sterile, 50/pkgindividually bagged, 100 mL capacity

4823 0.45 µm Supor membrane, white, gridded, sterile, 50/pkgindividually bagged, 100 mL capacity



*For use with ambient temperature samples only.

Microbiological Media (2 mL Plastic Ampoules)


4302 M-FC Broth with rosolic acid, Fecal Coliforms 50/pkg

4306 Pseudomonas Broth, Pseudomonas sp. 50/pkg

4307 Trypticase Soy Broth – USP, Total Bacteria 50/pkg

4352 HPC Media with TTC Indicator, Total Bacteria 50/pkg

68105 MF-Endo Broth, Total Coliforms 50/pkg

68106 M-TGE Broth, Total Bacteria 50/pkg

68107 M-Green YM Broth, yeast and mold 50/pkg

68108 KF-Streptococcal Broth, Fecal Streptococcus 50/pkg

68109 Orange Serum Broth, Lactobacillus sp. 50/pkg

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